1. Protein Structure and Function
Chapter 4
Protein Structure and Function

2. Proteins Make up about 15% of the cell Have many functions in the cell
Enzymes
Structural
Transport
Motor
Storage
Signaling
Receptors
Gene regulation
Special functions

3. Shape = Amino Acid Sequence
Proteins are made of 20 amino acids linked by peptide bonds
Polypeptide backbone is the repeating sequence of the N-C-C-N-C-C… in the peptide bond
The side chain or R group is not part of the backbone or the peptide bond

4. Polypeptide Backbone

5. Amino Acids NOTE: You need to know this table
Hydrophilic
Hydrophobic

6. Protein Folding
The peptide bond allows for rotation around it and therefore the protein can fold and orient the R groups in favorable positions
Weak non-covalent interactions will hold the protein in its functional shape – these are weak and will take many to hold the shape

7. Non-covalent Bonds in Proteins

8. Globular Proteins
The side chains will help determine the conformation in an aqueous solution

9. Hydrogen Bonds in Proteins
H-bonds form between 1) atoms involved in the peptide bond; 2) peptide bond atoms and R groups; 3) R groups

10. Protein Folding
Proteins shape is determined by the sequence of the amino acids
The final shape is called the conformation and has the lowest free energy possible
Denaturation is the process of unfolding the protein
Can be down with heat, pH or chemical compounds
In the chemical compound, can remove and have the protein renature or refold

11. Refolding
Molecular chaperones are small proteins that help guide the folding and can help keep the new protein from associating with the wrong partner

12. Protein Folding
2 regular folding patterns have been identified – formed between the bonds of the peptide backbone
-helix – protein turns like a spiral – fibrous proteins (hair, nails, horns)
-sheet – protein folds back on itself as in a ribbon –globular protein

13.  Sheets Core of many proteins is the  sheet
Form rigid structures with the H-bond
Can be of 2 types
Anti-parallel – run in an opposite direction of its neighbor (A)
Parallel – run in the same direction with longer looping sections between them (B)

14.  Helix Formed by a H-bond between every 4th peptide bond – C=O to N-H
Usually in proteins that span a membrane
The  helix can either coil to the right or the left
Can also coil around each other – coiled-coil shape – a framework for structural proteins such as nails and skin
 Helix

15. CD from Text
The CD that is included on your textbook back cover has some video clips that will show the  helix and  sheets as well as other things in this chapter. You will want to look at them. If you have problems, we will look at them during lab.

16. Levels of Organization
Primary structure
Amino acid sequence of the protein
Secondary structure
H bonds in the peptide chain backbone
-helix and -sheets
Tertiary structure
Non-covalent interactions between the R groups within the protein
Quanternary structure
Interaction between 2 polypeptide chains

17. Protein Structure

18. Domains
A domain is a basic structural unit of a protein structure – distinct from those that make up the conformations
Part of protein that can fold into a stable structure independently
Different domains can impart different functions to proteins
Proteins can have one to many domains depending on protein size

19. Domains

20. Useful Proteins
There are thousands and thousands of different combinations of amino acids that can make up proteins and that would increase if each one had multiple shapes
Proteins usually have only one useful conformation because otherwise it would not be efficient use of the energy available to the system
Natural selection has eliminated proteins that do not perform a specific function in the cell

21. Protein Families
Have similarities in amino acid sequence and 3-D structure
Have similar functions such as breakdown proteins but do it differently

22. Proteins – Multiple Peptides
Non-covalent bonds can form interactions between individual polypeptide chains
Binding site – where proteins interact with one another
Subunit – each polypeptide chain of large protein
Dimer – protein made of 2 subunits
Can be same subunit or different subunits

23. Single Subunit Proteins

24. Different Subunit Proteins
Hemoglobin
2  globin subunits
2  globin subunits

25. Protein Assemblies Proteins can form very large assemblies
Can form long chains if the protein has 2 binding sites – link together as a helix or a ring
Actin fibers in muscles and cytoskeleton – is made from thousands of actin molecules as a helical fiber

26. Types of Proteins
Globular Proteins – most of what we have dealt with so far
Compact shape like a ball with irregular surfaces
Enzymes are globular
Fibrous Proteins – usually span a long distance in the cell
3-D structure is usually long and rod shaped

27. Important Fibrous Proteins
Intermediate filaments of the cytoskeleton
Structural scaffold inside the cell
Keratin in hair, horns and nails
Extracellular matrix
Bind cells together to make tissues
Secreted from cells and assemble in long fibers
Collagen – fiber with a glycine every third amino acid in the protein
Elastin – unstructured fibers that gives tissue an elastic characteristic

28. Collagen and Elastin

29. Stabilizing Cross-Links
Cross linkages can be between 2 parts of a protein or between 2 subunits
Disulfide bonds (S-S) form between adjacent -SH groups on the amino acid cysteine

30. Proteins at Work
The conformation of a protein gives it a unique function
To work proteins must interact with other molecules, usually 1 or a few molecules from the thousands to 1 protein
Ligand – the molecule that a protein can bind
Binding site – part of the protein that interacts with the ligand
Consists of a cavity formed by a specific arrangement of amino acids

31. Ligand Binding

32. Formation of Binding Site
The binding site forms when amino acids from within the protein come together in the folding
The remaining sequences may play a role in regulating the protein’s activity

33. Antibody Family
A family of proteins that can be created to bind to almost any molecule
Antibodies (immunoglobulins) are made in response to a foreign molecule ie. bacteria, virus, pollen… called the antigen
Bind together tightly and therefore inactivates the antigen or marks it for destruction

34. Antibodies
Y-shaped molecules with 2 binding sites at the upper ends of the Y
The loops of polypeptides on the end of the binding site are what imparts the recognition of the antigen
Changes in the sequence of the loops make the antibody recognize different antigens - specificity

35. Antibodies

36. Binding Strength Can be measured directly
Antibodies and antigens are mixing around in a solution, eventually they will bump into each other in a way that the antigen sticks to the antibody, eventually they will separate due to the motion in the molecules
This process continues until the equilibrium is reached – number sticking is constant and number leaving is constant
This can be determined for any protein and its ligand

37. Equilibrium Constant
Concentration of antigen, antibody and antigen/antibody complex at equilibrium can be measured – equilibrium constant (K)
Larger the K the tighter the binding or the more non-covalent bonds that hold the 2 together

38. Enzymes as Catalysts
Enzymes are proteins that bind to their ligand as the 1st step in a process
An enzyme’s ligand is called a substrate
May be 1 or more molecules
Output of the reaction is called the product
Enzymes can repeat these steps many times and rapidly, called catalysts
Many different kinds – see table 5-2, p 168

39. Enzymes at Work
Lysozyme is an important enzyme that protects us from bacteria by making holes in the bacterial cell wall and causing it to break
Lysozyme adds H2O to the glycosidic bond in the cell wall
Lysozyme holds the polysaccharide in a position that allows the H2O to break the bond – this is the transition state – state between substrate and product
Active site is a special binding site in enzymes where the chemical reaction takes place

40. Lysozyme
Non-covalent bonds hold the polysaccharide in the active site until the reaction occurs

41. Features of Enzyme Catalysis

42. Enzyme Performance E + S  ES  EP  E + P
Step 1 – binding of the substrate
Limiting step depending on [S] and/or [E]
Vmax – maximum rate of the reaction
Turnover number determines how fast the substrate can be processed = rate of rxn  [E]
Step 2 – stabilize the transition state
State of substrate prior to becoming product
Enzymes lowers the energy of transition state and therefore accelerates the reaction

43. Reaction Rates
KM – [S] that allows rxn to proceed at ½ it maximum rate

44. Prosthetic Groups
Occasionally the sequence of the protein is not enough for the function of the protein
Some proteins require a non-protein molecule to enhance the performance of the protein
Hemoglobin requires heme (iron containing compound) to carry the O2
When a prosthetic group is required by an enzyme it is called a co-enzyme
Usually a metal or vitamin
These groups may be covalently or non-covalently linked to the protein

46. Regulation of Enzymes
Regulation of enzymatic pathways prevent the deletion of substrate
Regulation happens at the level of the enzyme in a pathway
Feedback inhibition is when the end product regulates the enzyme early in the pathway

47. Feedback Regulation
Negative feedback – pathway is inhibited by accumulation of final product
Positive feedback – a regulatory molecule stimulates the activity of the enzyme, usually between 2 pathways
 ADP levels cause the activation of the glycolysis pathway to make more ATP

48. Allostery
Conformational coupling of 2 widely separated binding sites must be responsible for regulation – active site recognizes substrate and 2nd site recognizes the regulatory molecule
Protein regulated this way undergoes allosteric transition or a conformational change
Protein regulated in this manner is an allosteric protein

49. Allosteric Regulation
Method of regulation is also used in other proteins besides enzymes
Receptors, structural and motor proteins

50. Allosteric Regulation
Enzyme is only partially active with sugar only but much more active with sugar and ADP present

51. Phosphorylation
Some proteins are regulated by the addition of a PO4 group that allows for the attraction of + charged side chains causing a conformation change
Reversible protein phosphorylations regulate many eukaryotic cell functions turning things on and off
Protein kinases add the PO4 and protein phosphatase remove them

52. Phosphorylation/Dephosphorylation
Kinases capable of putting the PO4 on 3 different amino acid residues
Have a –OH group on R group
Serine
Threonine
Tyrosine
Phosphatases that remove the PO4 may be specific for 1 or 2 reactions or many be non-specific

53. GTP-Binding Proteins (GTPases)
GTP does not release its PO4 group but rather the guanine part binds tightly to the protein and the protein is active
Hydrolysis of the GTP to GDP (by the protein itself) and now the protein is inactive
Also a family of proteins usually involved in cell signaling switching proteins on and off

54. Molecular Switches

55. Motor Proteins
Proteins can move in the cell, say up and down a DNA strand but with very little uniformity
Adding ligands to change the conformation is not enough to regulate this process
The hydrolysis of ATP can direct the the movement as well as make it unidirectional
The motor proteins that move things along the actin filaments or myosin

56. Protein Machines
Complexes of 10 or more proteins that work together such as DNA replication, RNA or protein synthesis, trans-membrane signaling etc.
Usually driven by ATP or GTP hydrolysis
See video clip on CD in book